1. Field of the Invention
The present invention relates to a method of regulating TGF-xcex2 activity. In particular, the present invention relates to a method of stimulating or inhibiting TGF-xcex2 activity by the application of specific peptides.
2. Background Art
Transforming growth factor-xcex2 (TGF-xcex2) is a member of a family of growth, differentiation, and morphogenesis autocrine and paracrine factors (3,26). TGF-xcex2 can affect diverse cellular functions in virtually all cell types. Depending on the cell type and its extracellular environment, these effects can be either positive or negative. TGF-xcex2 inhibits the proliferation of endothelial cells in vitro (31), but stimulates angiogenesis in vivo (39). TGF-xcex2 has also been shown to enhance or inhibit the proliferation of fibroblasts depending on the nature of the substrate and the mitogens present (3). Myoblast differentiation can also be induced or blocked by TGF-xcex2 depending on the availability of mitogens (25, 45).
TGF-xcex21 is a disulfide-linked homodimer that is synthesized as part of a latent precursor molecule (26). The latent precursor molecule is 390 amino acids in length and consists of an N-terminal 278 amino acid latency associated peptide (LAP) and a C-terminal 112 amino acid active domain (15-17). The proregion of TGF-xcex2 is unique in that it remains non-covalently attached to the active region after intracellular proteolytic processing and secretion (15). Association of the LAP with the mature peptide region confers latency: the LAP-associated growth factor is unable to interact with its cellular receptors. The LAP contains three N-linked glycosylation sites, two of which have mannose-6-phosphate residues (8,28,38). These carbohydrate structures may be important for latency since endoglycosidase F treatment leads to activation of TGF-xcex2 (28). The disulfide-bonded dimeric structure of LAP is critical for latency, since site-directed mutagenesis of critical cysteine residues (cys 223, 225) in the LAP abolishes the latency function (9). The active domain contains nine conserved cysteine residues that participate in inter-and intrachain disulfide bonding (27).
TGF-xcex2 is secreted by most cell types as a latent complex (27,37). Since TGF-xcex2 synthesis and TGF-xcex2 receptor expression are not highly regulated, primary regulation of TGF-xcex2 activity occurs by controlling conversion of the latent TGF-xcex2 complex to the active molecule. Physiochemical activation can occur by extremes of pH, heat, chaotropic agents, and deglycosylation (6,27,28,37). Activation in vivo is more complex and not well understood. There is evidence from cell culture models that activation may occur through binding of the latent molecule to mannose 6-phosphate receptors (12,21), by plasmin-mediated proteolytic processing (4,23,40,41), and/or by processing in acidic cellular microenvironments (20). In some systems, activation of latent TGF-xcex2 by plasmin is relatively inefficient (41). In addition, there are reports of TGF-xcex2 activation occurring independently of these mechanisms (19). These results suggest that additional mechanisms of latent TGF-xcex2 activation may exist.
TGF-xcex2 has been demonstrated, through numerous studies, to play a significant role in wound healing and fibrosis. The three phases of inflammation, granulation tissue formation and biosynthesis of the extracellular matrix, are identical in both the wound healing process and the development of fibrosis. A fine balance in the biosynthetic and degradative pathways involved in extracellular matrix biosynthesis appears to be determinative of whether proper wound healing or fibrosis results. Due to its function of regulating genes critically involved in extracellular matrix formation, TGF-xcex2 significantly influences this phase of tissue regeneration, the final outcome of which is either wound healing or fibrosis. (46). Thus, sensitive regulation of TGF-xcex2 activity in this process will permit control of the wound healing and fibrotic processes.
The thrombospondins (TSP) are a growing family of multidomain glycoproteins (47,48,5,49). TSP1 is the best characterized and serves as the prototypical TSP molecule. TSP is secreted and incorporated into the extracellular matrix of a number of cells in culture (1-5). TSP, like TGF-xcex2, has diverse effects on cellular functions that vary with cell type. TSP can inhibit endothelial proliferation and migration (2,42,34,51), but stimulates the growth of smooth muscle cells and dermal fibroblasts (52,36). TSP may also serve as both an attachment protein and an anti-adhesive molecule as shown by its ability to cause disassembly of focal adhesions in endothelial cells (33). TSP also plays a role in angiogenesis, fibrinolysis, platelet aggregation, and inflammation (1-5).
TSP is present transiently in wound environments and its synthesis is rapidly induced by growth factors, including TGF-xcex2 (50). TSP is detectable in incisional wound margins for 2-7 days, after which it localizes around vascular channels near the wound. Although the role of TSP is not yet clearly understood, it has been speculated that TSP may facilitate cell migration into the wound site or possibly act as a localized growth promoting agent (49).
There are three sequences in TSP known as type 1 repeats. Each repeat consists of approximately 60 amino acids, has six conserved cysteine residues and has approximately 47% sequence homology to similar repeats found in the human complement component, properdin. Within the type 1 repeats of TSP, there are two well defined consensus sequences, CSVTCG (SEQ ID NO:1) and WSXW (SEQ ID NO:2). CSVTCG (SEQ ID NO:1) inhibits metastasis of melanoma cells in a murine lung colonization assay (Tuszynski et al., 1992), and promotes cell adhesion possibly by mediating interactions of TSP with sulfated glycoconjugates (53,54). The anti-angiogenic activity of TSP might be, in part, due to CSVTCG (SEQ ID NO:1). Tolsma et al. showed that CSVTCG (SEQ ID NO:1) inhibits angiogenesis in vivo using a corneal neovascularization assay (55).
The sequence WSXW (SEQ ID NO:2) binds specifically to sulfated glycoconjugates and promotes cell adhesion and chemotaxis (56). The binding of TSP to the gelatin-binding domain of fibronectin can be blocked using the peptide GGWSHW (SEQ ID NO:3), suggesting WSHW (SEQ ID NO:4) may also promote matrix protein interactions (57). This sequence is also conserved within members of the TGF-xcex2 and cytokine receptor superfamilies (58,59).
This invention provides TSP peptides which activate latent TGF-xcex2 and TSP peptides which inhibit activation of latent TGF-xcex2.
TSP is a potential physiological regulator of TGF-xcex2 activity. These peptides from TSP can both positively and negatively modulate TGF-xcex2 levels at nanomolar to micromolar concentrations, and, therefore, can be used as therapeutic agents in vivo for the promotion of wound healing and inhibition of fibrosis.
The invention provides a method of stimulating TGF-xcex2 activity, comprising contacting latent TGF-xcex2 with an amount of an activating peptide effective to convert latent TGF-xcex2 to active TGF-xcex2. Also provided is a method of inhibiting the stimulation of TGF-xcex2 activity, comprising contacting latent TGF-xcex2 with an amount of an inhibiting peptide effective to inhibit the conversion of latent TGF-xcex2 to active TGF-xcex2.
The invention also provides a method of enhancing wound healing, comprising administering to the wound site an amount of an activating peptide effective to convert latent TGF-xcex2 to active TGF-xcex2, the activation of TGF-xcex2 resulting in enhanced wound healing.
A method of preventing fibrosis stimulated by TGF-xcex2 in pathology also provided. The method comprises administering to the site of potential fibrosis an amount of inhibiting peptide effective to inhibit conversion of latent TGF-xcex2 to active TGF-xcex2, resulting in reduced fibrosis.
The invention also provides a method of blocking TGF-xcex2-mediated inhibition of endothelial cell proliferation comprising contacting the endothelial cells with an inhibiting peptide effective to inhibit conversion of latent TGF-xcex2 to active TGF-xcex2, resulting in proliferation of endothelial cells.
The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included herein.
In one embodiment, the present invention provides a method of stimulating TGF-xcex2 activity, comprising contacting latent TGF-xcex2 with an amount of purified thrombospondin or a purified activating peptide effective to convert latent TGF-xcex2 to active TGF-xcex2. xe2x80x9cActivated TGF-xcex2xe2x80x9d or xe2x80x9cTGF-xcex2 activityxe2x80x9d as used herein describes the TGF-xcex2 protein present in a conformation whereby the TGF-xcex2 protein exerts an effect on cells to which it is exposed, the effect being proliferation, differentiation, angiogenesis, etc. xe2x80x9cLatent TGF-xcex2xe2x80x9d as used herein means the TGF-xcex2 protein present in a conformation whereby the active domain of the TGF-xcex2 protein is complexed to LAP and therefore, does not exert an effect on cells to which it is exposed. The term xe2x80x9cactivating peptidexe2x80x9d as used herein is defined as a peptide sequence or peptide mimetic, either synthetic, or generated from a native protein or by recombinant methods, comprising a minimum of three amino acids which, when exposed to latent TGF-xcex2, converts latent TGF-xcex2 to activated TGF-xcex2. The peptides of the invention correspond to sequences of TSP or they can be derived from the functional sequences of TSP. The term xe2x80x9cpurifiedxe2x80x9d as used herein means separated from other proteins, peptides and contaminants.
In the method of stimulating TGF-xcex2 activity, the activating peptide can be from the first, second and third type 1 repeat regions of TSP. For example, SEQ ID NOS: 5, 9, 14, 15 and 16 are from the second type 1 repeat region. As used herein, xe2x80x9csecond type 1 repeat regionxe2x80x9d means the second type 1 repeating sequence unit, as measured from the amino terminus of the three type 1 repeats and consisting of amino acids 412-473 of human TSP1. The activating peptide can be selected from the group consisting of: KRFK (SEQ ID NO:5), HRFK (SEQ ID NO:6), RKPK (SEQ ID NO:7), QRFK (SEQ ID NO:8), KRFKQDGG (SEQ ID NO:9), RWRPWTAWSE (SEQ ID NO:10), TAYRWRLSHRPKTGIRV (SEQ ID NO:11), KRFKQDGGASHASPASS (SEQ ID NO:12), KRFKQDGGASHASP (SEQ ID NO:13), KRFKQDGGWSHWSP (SEQ ID NO:14), KRFKQDGGWSHWSPWSS (SEQ ID NO:15), KRFKQDGGWSHW (SEQ ID NO:16) and KRFKQDGGWWSP (SEQ ID NO:17) or can consist of the amino acid sequence RFK (SEQ ID NO:18). The activating peptide may contain partial or full retro-inverso modifications of the sequences or appropriate non-natural amino acids. Such sequences as well as others corresponding to or derived from TSP are determined to be activating sequences by screening for TGF-xcex2 activating function in a soft agar NRK colony formation assay and an endothelial cell proliferation assay as described in the Examples.
In another embodiment, the invention provides a method of inhibiting the stimulation of TGF-xcex2 activity comprising contacting latent TGF-xcex2 with an amount of a purified inhibiting peptide effective to inhibit the conversion of latent TGF-xcex2 to activated TGF-xcex2. As used herein, xe2x80x9cinhibitory peptidexe2x80x9d means a peptide sequence comprising a minimum of four amino acids derived from the functional regions of TSP which, when exposed to latent TGF-xcex2, inhibits the conversion of latent TGF-xcex2 to active TGF-xcex2. The purified inhibiting peptide can have a sequence that corresponds to a sequence of four consecutive amino acids of TSP, effective to inhibit the conversion of latent TGF-xcex2 to active transforming TGF-xcex2.
In the method of inhibiting the stimulation of TGF-xcex2 activity, the inhibiting peptide can be from the first, second and third type 1 repeat region of TSP. For example, SEQ ID NOS: 25, 26, 27 and 3 are from the second type 1 repeat. The inhibiting peptide derived from TSP can consist of the amino acid sequence GGWSHW (SEQ ID NO:3) or selected from the group consisting of: WNDWI (SEQ ID NO:19), WSSWS (SEQ ID NO:20), LSKL (SEQ ID NO:21), AAWSHW (SEQ ID NO:22), DGWSPW (SEQ ID NO:23), GGWGPW (SEQ ID NO:24), WSPWS (SEQ ID NO:25), GWSHW (SEQ ID NO:26) and WSHWS (SEQ ID NO:27). The activating peptide may contain partial or full retro-inverso modifications of the sequences or appropriate non-natural amino acids. These and other sequences derived from TSP are determined to be inhibiting sequences by screening for inhibition of TGF-xcex2 activating function in a soft agar NRK colony formation assay as described in the Examples.
TGF-xcex2 is known to regulate wound healing. Thus, the present invention also provides a method of enhancing wound healing by administering to the wound site an amount of a purified activating peptide effective to convert latent TGF-xcex2 to active TGF-xcex2, the activation of TGF-xcex2 resulting in enhanced wound healing. The activating peptides can be those described herein. As used herein, xe2x80x9cenhanced wound healingxe2x80x9d is defined as a statistically significant increase in the rate of wound healing, as determined by histological analysis, tensile strength and total protein and collagen content of a wound treated with an activating peptide, as compared to a similar untreated wound or a similar wound treated with an inactive peptide control. Histological analysis includes examination for the presence of fibroblasts and capillary endothelial cells, which are early signs of wound healing. One example of this method, using the peptide KRFK (SEQ ID NO:5), is provided in the Examples.
In the method of enhancing wound healing, the activating peptide can be selected from the group consisting of: KRFK (SEQ ID NO:5), HRFK (SEQ ID NO:6), RKPK (SEQ ID NO:7), QRFK (SEQ ID NO:8), KRFKQDGG (SEQ ID NO:9), RWRPWTAWSE (SEQ ID NO:10), TAYRWRLSHRPKTGIRV (SEQ ID NO:11), KRFKQDGGASHASPASS (SEQ ID NO:12), KRFKQDGGASHASP (SEQ ID NO:13), KRFKQDGGWSHWSP (SEQ ID NO:14), KRFKQDGGWSHWSPWSS (SEQ ID NO:15), KRFKQDGGWSHW (SEQ ID NO:16) and KRFKQDGGWWSP (SEQ ID NO:17) or can consist of the amino acid sequence RFK (SEQ ID NO:18). Such sequences are determined to be activating sequences which enhance wound healing by screening for enhanced wound healing in rat models of wound healing as described in the Examples.
Because TGF-xcex2 plays a role in the development of fibrosis, the present invention also provides a method of preventing fibrosis stimulated by TGF-xcex2 in pathology by administering to the site of potential fibrosis an amount of a purified inhibiting peptide effective to inhibit conversion of latent TGF-xcex2 to active TGF-xcex2, resulting in reduced fibrosis. The inhibiting peptides can include those described herein. As used herein, xe2x80x9cfibrosisxe2x80x9d means the abnormal formation of fibrous tissue (60,64). xe2x80x9cReduced fibrosis,xe2x80x9d as used herein, is defined as the statistically significant reduction in the level of abnormal formation of fibrous tissue as determined by histological analysis, tensile strength and total protein and collagen content in a wound treated with an inhibitory peptide as compared to the level of abnormal formation of fibrous tissue in a similar untreated wound or a similar wound treated with a peptide having no activity under conditions such that fibrosis is expected to develop. One example of this method, using the TSP peptide GGWSHW (SEQ ID NO:3), is provided in the Examples.
In the method of preventing fibrosis stimulated by TGF-xcex2 in athology, the inhibiting peptide can also be selected from the group consisting of: WNDWI (SEQ ID NO:19), WSSWS (SEQ ID NO:20), LSKL (SEQ ID O:21), AAWSHW (SEQ ID NO:22), DGWSPW (SEQ ID NO:23), GGWGPW (SEQ ID NO:24), WSPWS (SEQ ID NO:25), GWSHW (SEQ ID NO:26) and WSHWS (SEQ ID NO:27). Such sequences are determined to be inhibiting sequences which prevent fibrosis by screening for prevention of fibrosis in rat models of fibrosis formation as described in the Examples.
Active TGF-xcex2 inhibits the proliferation of endothelial and epithelial cells. Thus, in another embodiment, the present invention provides a method of blocking the TGF-xcex2 mediated inhibition of endothelial or epithelial cell proliferation, comprising contacting the cells with a purified inhibiting peptide effective to inhibit conversion of latent TGF-xcex2 to active TGF-xcex2, resulting in proliferation of the cells. As used herein, xe2x80x9cproliferationxe2x80x9d means an increase in the number of cells.
In the method of blocking the TGF-xcex2-mediated inhibition of cell proliferation, the cells can be arterial endothelial cells. Other cells that can proliferate in response to this method are capillary endothelial cells. The inhibiting peptide can be selected from the group consisting of: WNDWI (SEQ ID NO:19), WSSWS (SEQ ID NO:20), LSKL (SEQ ID NO:21), AAWSHW (SEQ ID NO:22), DGWSPW (SEQ ID NO:23), GGWGPW (SEQ ID NO:24), WSPWS (SEQ ID NO:25), GWSHW (SEQ ID NO:26) and WSHWS (SEQ ID NO:27) or can consist of the amino acid sequence GGWSHW (SEQ ID NO:3). Such sequences are determined to be inhibiting sequences by screening for TGF-xcex2-mediated inhibition of cell proliferation, as described in the Examples.
The invention provides TGF-xcex2 activating and inhibiting peptides derived from the functional sequences of TSP. The present invention also provides a purified peptide having 3 to 30 amino acids, wherein the peptide comprises a subsequence R1-X1-X2-X3-R2, wherein X1 is selected from the group onsisting of Arg and Lys, X2 is selected from the group consisting of Pro and he, X3 is selected from the group consisting of Lys and Arg, R1 is H2, acyl, or a eptide from 1 to 26 amino acids, R2 is H, NH2, or a peptide of from 1 to 26 mino acids, and wherein the peptide converts latent TGF-xcex2 to active TGF-xcex2.
The purified peptide can be selected from the group consisting of: RFK (SEQ ID NO:18) KRFK (SEQ ID NO:5), HRFK (SEQ ID NO:6), RKPK (SEQ ID NO:7), QRFK (SEQ ID NO:8), KRFKQDGG (SEQ ID NO:9), TAYRWRLSHRPKTGIRV (SEQ ID NO:11), and KRFKQDGGASHASPASS (SEQ ID NO:12) or can consist of the amino acid sequence RWRPWTAWSE (SEQ ID NO:10).
The purified peptide can be conjugated to a water soluble polymer using standard protein conjugation protocols such as these described in Harlow and Lane (61). For example, suitable water soluble polymers include polysucrose, dextran, polyethylene glycol and polyvinyl alcohol.
The purified peptide can also be selected from the group consisting of partial and full retro-inverso peptide sequences. As used herein, xe2x80x9cpartial and full retro-inverso peptide sequencesxe2x80x9d means peptide sequences, determined to be either activating or inhibiting, which comprise some D-amino acids (partial) or consist entirely of D-amino acids (full), gem-diaminoalkyl residues, and alkylmalohyl residues. These can have unmodified termini, or can include appropriate alkyl, acyl, or amine substitutions to modify the charge of the terminal amino acid residues.
The present invention further provides purified peptides consisting of the amino acid sequences LSKL (SEQ ID NO:21) and acetyl-WHSWAA-NH2 (SEQ ID NO:28), and their partial and full retro-inverso peptide sequences.
Due to the relatively short half-life of peptides in vivo, the effects of modified peptides with longer half-lives can be examined. For example, the retro-inverso amino acid sequences (i.e., composed of D-amino acids) of the peptides described herein, such as the KRFKQDGGWSHWSPWSS (SEQ ID NO:15) and GGWSHW (SEQ ID NO:3) peptides, can be employed as described. These are expected to have a longer half life, because D-amino acids cannot be metabolized by cells as can naturally occurring L-forms of amino acids in proteins (65). Such retro-inverso peptides can be synthesized by standard peptide synthesis methods using commercially available D-amino acids (74). Peptide mimetics may be employed as substitutes for the natural peptide sequences based on established methods (75).
The described peptides can be applied in in vivo models to verify their modulation of TGF-xcex2-mediated effects of wound healing and fibrosis formation. For example, rat models of wound healing can be used to evaluate the effectiveness of KRFK (SEQ ID NO:5) in stimulating wound healing in comparison to active TGF-xcex2 (62,63). An inactive peptide can be used as a negative control (e.g., TRIR (SEQ ID NO:30), KRAK (SEQ ID NO:35)). The GGWSHW (SEQ ID NO:3) peptide or other inhibiting peptides provided herein can also be examined for any effect on inhibiting wound healing by blocking TGF-xcex2 activation or for any effect in keloid formation. Inactive analogues of this peptide can be used as negative controls. The in vivo protocol of Sporn et al. (62) can be used to determine the relative effectiveness of the activating peptides described herein on wound healing. For example, 2 cm by 1 cm wire mesh wound chambers can be implanted in the backs of rats. After a wound healing response is initiated (day 4), rats can be given daily injections of either 1000 ng TGF-xcex2, 100-1000 nM of activating peptides, 100-1000 nM TSP, 1000 ng albumin or vehicle control per injection site at the wound site. On day 9, the animals can be sacrificed and tissues in the wound chambers can be examined histologically, assayed for total protein and collagen content (by measurement of hydroxy-proline content) and relative levels of TGF-xcex2 in the wound tissue can be examined by immunohistochemical techniques.
Alternately, a rat model of incisional wound healing as descnbed by Cromack et al. (63), can be used. In this system, a 6 cm linear incision can be made on the dorsal skin of a rat, the wound can be coapted with surgical clamps and 100-100 nM of activating peptides can be injected at the wound site in 3% methylcellulose as a vehicle. After 7-10 days, the wound strips can be harvested and evaluated for tensile strength using a tensiometer and for histological analysis as described herein.
The above-descnbed protocols can be applied to humans, because wound healing and fibrosis formation in rats, rabbits and pigs are commonly used as models for the study of wound healing and fibrosis formation in humans. (66-69).
In a clinical application, 1 xcexcg to 100 mg of the activating peptides can be used to impregnate bandages or as part of an ointment to be applied to wound areas for the purpose of enhancing wound healing or preventing fibrosis. A skilled clinician would be able to determine, more specifically, the amount of peptides and length of treatment necessary to enhance wound healing or inhibit fibrosis.
The present peptides may be administered parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like, although topical administration is typically preferred. The exact amount of such compounds required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the wound or disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using methods well known in the art.
For topical administration, the compounds of the present invention can be in pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example powders, liquids, suspension, lotions, creams, gels or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Parenteral administration can also employ the use of a slow release or sustained release system, such that a constant level of dosage is maintained (See, for example, U.S. Pat. No. 3,710,795).
Utility
The present invention also provides a bioassay for screening substances for their ability to modulate the activity of thrombospondin. For example, the present invention provides a bioassay for screening substances for their ability to enhance the activity of thrombospondin, for example, for use in therapies to promote wound healing. In another example, the present invention provides a bioassay for screening substances for their ability to inhibit the activity of TSP, for example, for use in therapies for prevention of fibrosis. Briefly, these bioassays can be performed in vitro by administering a substance to NRK cells with TSP peptides and latent TGF-xcex2 and assaying for soft agar colony formation as descnbed in the Examples. Alternatively, these bioassays can be performed in vitro by administering a substance to BAE cells and measuring cell proliferation as described in the Examples. The current use of such screening methods is set forth in the Examples, which were used to show that the peptide KRFK (SEQ ID NO:5) activates TGF-xcex2 and the peptide GGWSHW (SEQ ID NO:3) inhibits TSP-mediated activation of latent TGF-xcex2. In vivo, these bioassays can be performed by administering substances to the wound chambers and wound sites as descnbed herein to screen for substances which play a role in wound healing and fibrosis.
The present invention further provides peptides which activate and inhibit TGF-xcex2, which can be used as controls in in vitro bioassays for screening substances for their ability to modulate the activity of TGF-xcex2. For example, the substances and TGF-xcex2 can be administered to NRK cells which can then be assayed for soft agar colony formation as described in the Examples. Activating or inhibiting peptides can be administered to NRK cells which can then be assayed for soft agar colony formation as positive controls for TGF-xcex2 activation and inhibition. These activating and inhibiting peptides can also be used as controls in in vivo bioassays for screening substances for their abililty to modulate the activity of TGF-xcex2. For example, substances can be applied to the wounds and sites of potential fibrosis in the assays described in the Examples and evaluated for their ability to enhance wound healing and reduce fibrosis. The activating and inhibiting peptides can be used as controls in the described Examples.
The invention provides a method of generating a purified antibody specifically reactive with a peptide of the invention. The antibodies made can be used to detect the presence of the TSP protein. Antibodies can be made as descnbed in the art (61). Briefly, purified peptide alone or peptide conjugated to a carrier protein can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or, for monoclonal antibodies, spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion.
The following examples are intended to illustrate, but not limit, the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may be alternatively employed.